The cullin-RING ubiquitin ligase (CRL) family contains upwards of 240 ubiquitin ligases that regulate a broad range of cellular and organismal processes. Consistent with their critical roles in regulatory biology, CRLs have been intimately linked to human disease pathogenesis and therapy: mutations in CRL subunits underlie the development of several human cancers and CRL4CRBN has been implicated as the target of the anti- myeloma agent Thalomid. CRL pathways are regulated by multiple mechanisms, including dynamic cycles of CRL assembly/disassembly and post-translational modification of the cullin subunit by the ubiquitin-like protein Nedd8. CSN is a Nedd8 isopeptidase that regulates CRLs by removing the Nedd8 modification. Removal of Nedd8 from a CRL results in its deactivation and renders its substrate receptor susceptible to disassembly by the exchange factor, Cand1. Although the isopeptidase activity of CSN inhibits CRL activity, genetic studies indicate that CSN is a positive regulator of CRLs, which suggests that periodic deactivation and disassembly of CRLs is required to sustain CRL activity. Recently, the Nedd8 conjugation pathway has emerged as a drug target for cancer chemotherapy and the active site subunit of Csn5 has been implicated in sustaining tumorigenicity in a Myc-driven cancer model. Together, these data point to dynamic cycling of CRLs as being critical for cancer cell proliferation. The over-arching hypotheses that animate this project are that CSN plays a critical role in regulating the activity of CRL enzymes and architecture of the CRL network, and that perturbation of CSN activity is toxic to cancer cells. We plan to explore these topics in three Specific Aims. In Aim 1 we will use quantitative multidimensional mass spectrometry to characterize the architecture and dynamics of the CRL proteome. In Aim 2, we will characterize a novel non-enzymatic CRL-inhibitory activity of CSN. In Aim 3, we will generate a CSN isopeptidase inhibitor to use as a research tool and to evaluate whether CSN is a promising target for chemotherapy of cancer.